1. Field of Invention
The present invention relates to a type IV collagen high molecular form and to a process for its production. The invention further relates to a method for measuring the type IV collagen high molecular form and to a method of diagnosing the degree of liver fibrosis based on that assay.
2. Related Art
It is well known that liver fibrosis occurs in patients with liver diseases, and is caused by the unbalance between synthesis and degradation of type IV collagen in the liver, leading to excess synthesis rather than degradation. A number of reports have been published on the usefulness of serum type IV collagen determination as an indicator of liver fibrosis, and it is known that type IV collagen level in the serum increases with progression of diseases from chronic hepatitis to liver cirrhosis (Japanese Unexamined Patent Publication No. 8-100000, Saito et al., Liver, Vol.37, No.6, p.304-311). This occurs because the major component of fibrotic regions in the liver is type IV collagen, and therefore serum levels of type IV collagen directly reflect the histological degree of liver fibrosis.
Incidentally, the type IV collagen molecules in the blood are released into the blood during the progression of liver fibrosis, i.e. during the process of synthesis and degradation of type IV collagen in the liver, and they are not uniform in terms of molecular species. This is because the structure of type IV collagen is a network structure composed of the amino terminal 7S domain, the carboxyl terminal NC1 and NC2 regions and an intermediate triple helical region which come together in a helix form, and this structure is partially degradated by collagenase and released into the blood.
According to reports by Murawaki et al. in J. Hepatology, Vol.24, p.148-154, 1996 and Clinica Chimica Acta, 212, p.73-78, (1992) (hereunder, "C.C.A"), when an assay is performed using an antibody which recognizes the 7S domain, primarily 2 major molecular species are detected as type IV collagen in the blood: (1) a high molecular form containing the 7S domain and having a molecular weight higher than that of the 7S domain, and (2) the 7S domain itself. According to the same reports, when the assay is performed using an antibody which recognizes the 7S domain and an antibody which recognizes the triple helical region, a type IV collagen smaller than both (1) and (2) is detected. These facts demonstrate that 3 kind of molecules which have different molecular weights exist as the type IV collagen molecules in serum.
Reports in C.C.A also indicate that these 3 molecular species are present as type IV collagen in the blood of both healthy persons and patients with liver disease. However, during the progression of diseases from chronic hepatitis to liver cirrhosis in patients, the serum level of the high molecular form which contains the 7S domain and has a larger size than that of 7S domain, is notably increased among the 3 molecular species of type IV collagen. This type IV collagen high molecular form is believed to be type IV collagen in almost complete form without any degradation by collagenase, and therefore increases or decreases in the amount of the high molecular form of type IV collagen in the blood are considered to be direct reflections of the increase or decrease in type IV collagen synthesis, while the increase or decrease in the 7S domain or lower molecular size type IV collagen in the blood are considered to reflect the increase or decrease in type IV collagen degradation from fibrotic liver.
However, until now there have been no reports regarding separation and purification of the high molecular form of type IV collagen. Even in the aforementioned reports by Murawaki et al., where the patient serum is separated by gel filtration and a commercially available type IV collagen assay kit is used to determine the reactivity of each fraction, it is merely indicated that antigenicity is also found in the fraction having a higher molecular weight than that of 7S domain.
Although commercially available type IV collagen-assay kits exist which can show the reactivity with the type IV collagen high molecular form, they have high reactivity with the aforementioned 7S domain itself or for the smaller type IV collagen, whereas no kit has been available which allows specific determination of only the type IV collagen high molecular form. Consequently, no assay method has existed which directly reflects the synthesis of type IV collagen in the liver.
Incidentally, a monoclonal antibody (hereunder referred to as "monoclonal antibody 67") which is produced by the hybridoma COL IV-67 (deposited at the National Institute of Bioscience and Human Technology on Sep. 27, 1994 as FERM P-14561, and transferred on Sep. 25, 1995 to a deposit in accordance with the Budapest Treaty, as FERM BP-5240) described in Japanese Unexamined Patent Publication No. 8-100000, has been shown to react specifically with the type IV collagen high molecular form, therefore it was assumed that the type IV collagen high molecular form was present in the immunogen for the monoclonal antibody 67, but the type IV collagen high molecular form could not be consistently obtained by the purification method for the immunogen described in Japanese Unexamined Patent Publication No. 8-100000.